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Abstract:

Disclosed is a composition for inducing the proliferation of retinal
cells or the differentiation of retinal progenitor cells into retinal
cells. The composition, similar to in vivo conditions for development
during embryogenesis, induces stem cells to differentiate into a
multitude of photoreceptor cells at high yield within a short period of
time, without an additional gene transfer. In addition, the
differentiated photoreceptor cells are useful in cellular therapy because
they, when transplanted into degenerated or injured retinas, can be
engrafted and fused within the retinas to prevent or cure retinal
degeneration.

Claims:

1. A method for inducing proliferation of retinal cells, comprising the
step of culturing the retinal cells in a medium containing a Wnt
signaling pathway activator.

2. The method as set forth in claim 1, wherein the retinal cells are
photoreceptor cells.

3. The method as set forth in claim 1, wherein the retinal cells are
human-derived cells.

8. The method as set forth in claim 7, wherein the GSK3 inhibitor is
selected from the group consisting of LiCl, BIO
(6-bromoindirubin-3'-oxime), SB415286 and a combination thereof.

9. The method as set forth in claim 4, wherein the concentration of the
Wnt signaling pathway activator used in the medium is ranging from 0.01
to 500 ng/ml.

10. The method as set forth in claim 9, the concentration of the Wnt
signaling pathway activator used in the medium is ranging from 1 to 100
ng/ml.

11. The method as set forth in claim 7, wherein the concentration of the
GSK inhibitor except for LiCl, BIO and SB415286 used in the medium is
ranging from 0.01 to 500 ng/ml.

12. The method as set forth in claim 11, wherein the concentration of the
GSK inhibitor except for LiCl, BIO and SB415286 used in the medium is
ranging from 1 to 100 ng/ml.

13. The method as set forth in claim 7, wherein the concentration of LiCl
used in the medium is ranging from 0.1 to 50 mM; that of BIO is ranging
from 0.1 to 50 μM; and that of SB415286 is ranging from 0.1 to 500
μM.

14. The method as set forth in claim 13, wherein the concentration of
LiCl used in the medium is ranging from 1 to 10 mM; that of BIO is
ranging from 0.5 to 5 μM; and that of SB415286 is ranging from 5 to 50
μM.

15. A method for inducing differentiation from retinal progenitor cells
into retinal cells, comprising the step of culturing the retinal
progenitor cells in a medium containing a Wnt signaling pathway
activator.

16. The method as set forth in claim 15, wherein the retinal cells are
photoreceptor cells.

17. The method as set forth in claim 15, which comprises: (a) culturing
the retinal progenitor cells in a medium containing a Wnt signaling
pathway activator to differentiate them into neural retinal progenitor
cells; (b) culturing the neural retinal progenitor cells of step (a) in a
medium containing a Wnt signaling pathway activator to differentiate them
into photoreceptor cell precursors; and (c) culturing the photoreceptor
cell precursors of step (b) in a medium containing a Wnt signaling
pathway activator to differentiate them into photoreceptor cells or
retinal cells including photoreceptor cells.

22. The method as set forth in claim 21, wherein the GSK3 inhibitor is
selected from the group consisting of LiCl, BIO
(6-bromoindirubin-3'-oxime), SB415286 and a combination thereof.

23. The method as set forth in claim 15, wherein the concentration of the
Wnt signaling pathway activator used in the medium is ranging from 0.01
to 500 ng/ml.

24. The method as set forth in claim 23, the concentration of the Wnt
signaling pathway activator used in the medium is ranging from 1 to 100
ng/ml.

25. The method as set forth in claim 21, wherein the concentration of the
GSK inhibitor except for LiCl, BIO and SB415286 used in the medium is
ranging from 0.01 to 500 ng/ml.

26. The method as set forth in claim 25, wherein the concentration of the
GSK inhibitor except for LiCl, BIO and SB415286 used in the medium is
ranging from 1 to 100 ng/ml.

27. The method as set forth in claim 21, wherein the concentration of
LiCl used in the medium is ranging from 0.1 to 50 mM; that of BIO is
ranging from 0.1 to 50 μM; and that of SB415286 is ranging from 0.1 to
500 μM.

28. The method as set forth in claim 27, wherein the concentration of
LiCl used in the medium is ranging from 1 to 10 mM; that of BIO is
ranging from 0.5 to 5 μM; and that of SB415286 is ranging from 5 to 50
μM.

Description:

TECHNICAL FIELD

[0001] The present invention relates to compositions for inducing the
proliferation of retinal cells and the differentiation of retinal
progenitor cells into retinal cells. More particularly, the present
invention relates to compositions for inducing the differentiation of
retinal progenitor cells into retinal cells, especially photoreceptor
cells and the proliferation of photoreceptor cells that comprise a Wnt
signaling pathway activator.

BACKGROUND ART

[0002] Blindness is the medical condition of lacking visual perception for
physiological or neurological reasons. As many as tens of millions of
people, which accounts for 0.2-0.5% of the population of the world, are
affected with blindness, and are suffering from great losses in personal,
social and economical respects. Retinal photoreceptor degeneration is one
of the more dominant etiologies of blindness, caused innately or by other
various factors, including retinal dysplasia, retinal degeneration, aged
macular degeneration, diabetic retinopathy, retinitis pigmentosa,
congenital retinal dystrophy, Leber congenital amaurosis, retinal
detachment, glaucoma, optic neuropathy, and trauma. No drugs have been
developed for the fundamental treatment of these diseases thus far. To
date, the replacement of dysfunctional photoreceptor cells, the alpha and
omega of these retinal diseases, with new ones is regarded as the only
promising therapy. Photoreceptor cell implantation is thought to prevent
blindness or recover imperfect eyesight by delaying or restraining
retinal degeneration, regenerating degenerated retina, and enhancing
retinal functions.

[0004] No significant research results have been yet suggested regarding
the differentiation of stem cells into retinal cells (particularly,
photoreceptor cells) and cell therapy based thereon. The differentiation
of these stem cells into retinal cells might make it possible 1) to
guarantee an infinite cell source for efficient cell therapy, 2) to
identify the differentiation mechanism from embryonic cells and retinal
progenitors into retinal cells, which has remained unclear, 3) to find
retina differentiation-related genes and molecules and lesions thereby,
4) to understand the pathogenesis of retinal degenerative diseases, and
5) to develop drugs for preventing retinal degeneration and protecting
the retina.

[0005] Since the first establishment thereof, human embryonic stem cell
lines have been suggested to have the ability to differentiate into
various types of cells which are useful for the cellular therapy of
various diseases. Human embryonic stem cells appear to have a high
potential when it comes to allowing the accurate examination of
pathogenetic mechanisms and supplying fresh cells that can substitute for
dysfunctional cells in clinical treatment. The production of human ESC
derived-retinal photoreceptor cells under a completely identified
reproducible condition and the use thereof in transplantation would
guarantee a highly potential and effective therapy for retinal
photoreceptor cell-related diseases. It has been assumed that human ESC
derived-cells will have the same properties and functions as did the
cells formed that were formed through a normal differentiation processes.
Based on this assumption, differentiation has been induced under
circumstances similar to those of the developmental stages to produce
pancreatic hormone-expressing endocrine cells (D'Amour, et al., Nat.
Biotechnol., 2006; 24: 1392-401), neurons (Pankratz, et al., Stem Cells
2007; 25: 1511-20), muscle cells (Barberi et al., Nat. Med., 2007; 13:
642-8), and vascular endothelial cells (Wang, et al., Nat. Biotechnol.,
2007; 25: 317-8). Also, many attempts have been made to differentiate
human ESC into photoreceptor cells which may be effectively used to treat
retinal diseases, but this ended with failure for most cases.

[0006] In fact, differentiation into retinal progenitor cells from human
embryonic stem cells is the greatest achievement made thus far in this
field, but the differentiation of retinal progenitor cells into
photoreceptor cells failed (differentiation rate of less than 0.01%)
(Lamba et al., Proc. Natl. Acad. Sci. USA, 2006; 103: 12769-74). One
report held that human embryonic stem cells were successfully induced to
differentiate into photoreceptor cells, but the method used therein
requires more than 200 days in total for the differentiation, with a
differentiation rate of as low as 8%, and thus is impossible to apply to
the clinical treatment of blindness (Osakada et al., Nat. Biotechnol.,
2008; 26: 215-24).

[0007] The Wnt signaling pathway participates in regulating various
processes during embryogenesis, including tissue development, cell
proliferation, morphology, motility and cell-fate determination, etc
(Wodarz & Nusse, Annu. Rev. Cell Dev. Biol., 1998; 14: 59-88). It is
known to promote or regulate differentiation depending on tissue type and
differentiation level. To date, in the context of embryonic development
and cell biology, the Wnt signaling pathway including Wnt3a has been
reported to be deeply involved in the regulation of cellular
differentiation and the maintenance and proliferation of undifferentiated
cells (Aubert, et al., Nat. Biotechnol., 2002; 20: 1240-5). Nowhere has
the effect of the Wnt signaling pathway on cell differentiation and
maturation associated with vertebrate eye patterning and neurogenesis
been reported in the prior art.

DISCLOSURE

Technical Problem

[0008] Leading to the present invention, intensive and thorough research
into the differentiation of human ESC into retinal cells, particularly
photoreceptor cells, conducted by the present inventors, using chemically
defined, resulted in the finding that when in vitro conditions for
differentiation into photoreceptor cells, similar to in vivo conditions,
in which differentiation-associated factors and their inhibitors were
employed, was applied, the Wnt signaling pathway activators played an
important role in the proliferation of retinal cells and the
differentiation and maturation from stem cells into retinal cells,
particularly photoreceptor cells.

Technical Solution

[0009] It is therefore an object of the present invention to provide a
composition for inducing the proliferation of retinal cells, comprising a
Wnt signaling pathway activator. It is another object of the present
invention to provide a composition for inducing differentiation from
retinal progenitor cells into retinal cells, particularly photoreceptor
cells, comprising a Wnt signaling pathway activator.

Advantageous Effects

[0010] As described above, the composition of the present invention allows
stem cells to differentiate into a multitude of photoreceptor cells at
high yield within a short period of time, without an additional gene
transfer. Able to generating a multitude of photoreceptor cells at high
yield, the composition of the present invention does neither require the
isolation of photoreceptor cells through flow cytometry nor an additional
photoreceptor proliferation so that the differentiated photoreceptor
cells may be readily transplanted into degenerated or injured retinas. In
addition, the differentiated photoreceptor cells are useful in cellular
therapy because they, when transplanted into degenerated or injured
retinas, can be engrafted and fused within the retinas to prevent or cure
retinal degeneration.

[0012] (A) Left. Typical cell floc of hESCs in an undifferentiated state
(29 passages; 40× magnification), after being cultured for 5 days
from cells of passage number 28. Characterized by a definite separation
from adjacent MEF feeder cells. Having plain surface and uniform
morphology.

[0015] (B). Cells on Day 14 after induction of the differentiation, that
is, after the floating aggregates were transferred to
poly-D-lysine/laminin-coated plates and cultured for 10 days therein,
which was on Day 14 after the induction of the differentiation of the
undifferentiated hESCs. The cells were observed to be separated from the
floating aggregates and to undergo differentiation. Morphology of cells
in the early stage of differentiation, with meager cytoplasm and round,
large nuclei.

[0016] (C). Cells on Day 19 after the induction of the differentiation,
that is, cells after the undifferentiated hESCs were induced to
differentiate for 19 days. They differentiated into retinal progenitor
cells, with concomitant active proliferation. Cell flocs under active
proliferation and differentiation formed an eddy formation or a rosette
configuration.

[0017] (D). Cells on Day 21 after induction of the differentiation,
showing an increased number of cells resulting from active proliferation.
The cells became richer in cytoplasm and the size of their nucleus was
smaller than those of FIG. 1C as the differentiation progressed. The
cells appeared to function in response to light.

[0018] (E)-(H). Various morphologies of cell flocs on Day 29 after
induction of the differentiation.

[0019] (E). The morphology of most cells, particularly observed in densely
populated regions. With the progress of differentiation, the cells showed
the same cellularity, but had a richer cytoplasm and smaller and denser
nuclei, compared to those on Day 21 after induction.

[0020] (F). The morphology of cells in a scarcely populated region. The
cell flocs showed directivity and moved towards a certain point which
depended on the cell cluster. More plentiful, opposite end-pointed
cytoplasms and spindle-like nuclei were observed.

[0025]FIG. 2 is a graph showing changes in expression levels of the
retinal cell markers Crx, recoverin, rhodopsin, peripherin2 and Ki67 with
culture time period.

[0026]FIG. 3 is a set of microphotographs showing the cells obtained by
differentiation into retinal cells for 29 days, which were immunostained
for recoverin and rhodopsin, both indicative of photoreceptor cells.

[0027] After hESCs were induced to differentiate into photoreceptor cells,
an examination was made of the expression of photoreceptor cell-specific
proteins. More than 80% of the differentiated cells tested positive to
both recoverin (a universal photoreceptor cell marker) and rhodopsin
(characteristic of rod photoreceptor cells).

[0028] (A) and (B). Flocs of differentiated photoreceptor cells.

[0029] (C). Individual cells in scarcely populated regions.

[0030] Recoverin and rhodopsin are distinctively expressed in the
differentiated photoreceptor cells. [0031] Microscopic field: (A)
100× magnification; (B) 200× magnification; (C) 400×
magnification. [0032] Merge: superimposed photographs of cells which were
fluorescent immunostained for recoverin and rhodopsin, cells expressing
both the antigens being represented yellow (green+red). [0033]+Merge/DAPI: DAPI is a nucleus-stained cell population. The
Merge/DAPI images are of superimposed fluorescence photographs to detect
the expression of both recoverin and rhodopsin and the expression of
DAPI, showing cell contours and the expression pattern of the two
antigens, simultaneously.

[0034] FIG. 4 is of fluorescence microphotographs of the cells obtained
after inducing differentiation into retinal cells for 29 days, showing
the expression of the photoreceptor cell markers rhodopsin, rom-1 and
peripherin2.

[0035] The differentiated photoreceptor cells were observed to express
Rom-1 and peripherin2, both characteristic of the outer segment of
rhodopsin-positive rod photoreceptor cells.

[0036] (A). Cell flocs positive to both rhodopsin and rom-1.

[0037] (B). Individual cells positive to both rhodopsin and rom-1. Within
each cell, rhodopsin and rom-1 were expressed at distinctly different
positions. With the progress of differentiation, rhodopsin was expressed
in the inner cytoplasm while rom-1 was positioned at the outermost
cytoplasm.

[0038] (C). Flocs of the differentiated photoreceptor cells which were
positive to both rhodopsin and peripherin2.

[0041] FIG. 5 is of fluorescence microphotographs of the cells obtained
after inducing differentiation into retinal cells for 29 days, showing
the expression of the photoreceptor cell markers rhodopsin, phosducin and
Pde6b. These proteins are responsible for the response to light,
demonstrating that the differentiated photoreceptor cells are exhibiting
their proper functions.

[0042] (A). Flocs of the differentiated photoreceptor cells which are
positive to both rhodopsin and phosducin.

[0043] (B). Individual cells positive to both rhodopsin and phosducin.

[0044] (C). Flocs of the differentiated photoreceptor cells positive to
both rhodopsin and Pde6b.

[0047]FIG. 6 is of fluorescence microphotographs of cells obtained after
inducing differentiation into retinal cells for 29 days, showing the
expression of the photoreceptor cell markers rhodopsin and synaptophysin.

[0048] (A). Flocs of the differentiated photoreceptor cells positive to
both rhodopsin and synaptophysin. The expression of these proteins
demonstrates that the differentiated photoreceptor cells are in synapse
interaction with other retinal neurons and are participating in the
formation of retinal nerve circuits.

[0056]FIG. 8 is of fluorescence microphotographs of cells which were
obtained after inducing differentiation into retinal cells for 29 days
and which were immunostained against characteristic photoreceptor cells.
Various types of cells which had undergone further differentiation were
observed.

[0057] Left. Cells positive to both recoverin and rhodopsin, showing a
morphology characteristic of photoreceptor cells.

[0061]FIG. 9 is of fluorescence microphotographs of cells which were
obtained after inducing differentiation into retinal cells for 29 days
and which were immunostained against neural retinal progenitor cells and
photoreceptor cell precursors.

[0062] (A). Cell flocs positive to both Rax and Pax6.

[0063] (B). Individual cells positive to both Rax and Pax6.

[0064] Most cells were observed to express both the antigens although the
expression level was different between them, indicating that the retinal
cells obtained after differentiation induction for 29 days were derived
from neural retinal progenitor cells.

[0069] FIG. 10 is of fluorescence microphotographs of cells which were
obtained after inducing differentiation into retinal cells for 29 days
and immunostained against retinal cells other than photoreceptor cells.

[0070] (A). Cell flocs (left) and individual cells, positive to both
Islet-1 and NF-200, which gives evidence of retinal ganglion cells
because nuclei and axons are positive to Islet-1 and NF-200,
respectively.

[0076] FIG. 11 is of fluorescence microphotographs of the cells resulting
from inducing differentiation into retinal cells for 29 days, with BIO
and purmorphamine used respectively instead of Wnt3a and Shh, which were
immunostained against photoreceptor cell precursors and photoreceptor
cells.

[0082]FIG. 12 is of fluorescence microphotographs of the cells resulting
from inducing differentiation into retinal cells for 29 days, with BIO
and purmorphamine used respectively instead of Want3a and Shh, which were
immunostained for the photoreceptor cell markers rhodopsin, peripherin2
and rom-1.

[0083] (A). Cell flocs positive to both rhodopsin and peripherin2.

[0084] (B). Individual cells positive to both rhodopsin and peripherin2.

[0088]FIG. 13 is of fluorescence microphotographs of the cells resulting
from inducing differentiation into retinal cells for 29 days, with BIO
and purmorphamine used respectively instead of Want3a and Shh, which were
immunostained against cone photoreceptor cells.

[0093] (A). Cytomorphological microphotographs. (A) Left. Typical cell
floc of human iPSCs in an undifferentiated state (passages 43;
Microscopic field: 40 magnifications), after being cultured for 6 days
from cells of passages 43. Characterized by definite separation from
adjacent MEF feeder cells. Having a plain surface and uniform morphology,
which is also characteristic of undifferentiated hESCs. (A) Right.
Floating aggregates (Microscopic field: 40× magnification), being
cultured for 4 days in ultra-low attachment plates after isolation from
the human iPSC floc of FIG. 14A Left.

[0094] (B). Fluorescence microphotographs of undifferentiated human iPSCs
immunostained for characteristic markers. Cell flocs in which most cells
are positive to both SSEA4 and Nanog, which gives evidence of the
continuance of undifferentiated states. [0095] Microscopic field
(leftmost) 40× magnification; (the others) 100×
magnification.

[0096] FIG. 15 is a set of fluorescence microphotographs showing the cells
obtained by inducing human iPSC to differentiate into retinal cells for
29 days, which were immunostained for recoverin and rhodopsin, both
characteristic of photoreceptor cells. The photoreceptor cells
differentiated from human iPSCs were assayed for the expression of
photoreceptor cell-specific proteins.

[0102]FIG. 16 is of photographs showing RT-PCR for genes specific for
retinal cells. The cells generated by inducing undifferentiated hESCs to
differentiate into retinal cells for 29 days were assayed for the mRNA
expression levels of genes associated with retinal progenitor cells,
photoreceptor cells and other retinal cells using RT-PCR.

[0105] Characteristics of photoreceptor cell-relevant genes are as
follows: CRX and NRL are transcription genes characteristic of
photoreceptor cell precursors and rod photoreceptor cells, respectively.
RCVRN (recoverin) is a universal photoreceptor cell gene that tests
positive for both cone and rod photoreceptor cells. RHO (rhodopsin) is
rod photoreceptor cell specific. PDE6B and SAG (human arrestin) are
involved in the phototransduction of photoreceptor cells. The expression
of these genes gives evidence of the development and maturation of the
photoreceptor cell's own functions. OPN1SW is characteristic of short
wave (blue opsin)-cone photoreceptor cells. M: marker.

[0107] RT-PCR was performed on the cells generated by inducing
undifferentiated hESCs to differentiate into retinal cells for 29 days,
so that the photoreceptor cell-specific genes RCVRN (NM--002903.2)
and RHO (NM--000539.3) could be detected. The RT-PCR products were
identified as RCVRN and RHO by base sequencing.

[0110]FIG. 18 is of electroretinograms of the retinal degeneration mouse
rd/SCID which had been or had not been transplanted with the hESC-derived
photoreceptor cells.

[0111] (A). Electroretinograms of 8-week-old, non-transplanted mice. No
characteristic ERG wave forms were found. The ERG b-wave had an amplitude
of 6.29 μV for the right eye and 0.0542 μV for the left eye.

[0112] (B). Electroretinograms of 8-week-old mice 4 weeks after the
transplantation. Compared to the non-transplanted right eye, the EGR
b-wave from the photoreceptor cell-transplanted left eye formed
characteristic wave forms, with an amplitude of as high as 74.5 μV.
The rd/SCID mice transplanted with the hESC-derived photoreceptor cells
showed definite responses to light stimuli as measured by
electroretinography.

[0113]FIG. 19 is a graph comparing the amplitude of the b-wave between
rd/SCID mice with retinal degeneration which had been transplanted or had
not been transplanted with the hESC-derived photoreceptor cells.

[0114] The ERG b-wave from the photoreceptor cell-transplanted rd/SCID
mice formed characteristic wave forms, with an amplitude of 48.4(±3.4)
μV (sample size=13). In contrast, characteristic wave forms where
nowhere to be found in the ERG of the non-transplanted group, which
showed a b-wave amplitude of 10.3(±2.5) μV (sample size=17) which
is different from that of the transplanted group with statistical
significance (p<0.0001) (Table 6, FIG. 19).

[0115]FIG. 20 is of fluorescence microphotographs after the hESC-derived
photoreceptor cells had been transplanted into the mouse model of retinal
degeneration (rd/SCID).

[0116] Four weeks after the transplantation, the hESC-derived
photoreceptor cells were analyzed for engraftment into the retina using
rhodopsin and recoverin, both characteristic of human mitochondria and
photoreceptor cells. When cells positive to rhodopsin and recoverin
showed a positive response to a human mitochondrial antigen, they were
decided to be hESC-derived photoreceptor cells.

[0117] (A). Immunostained human-specific mitochondria and rhodopsin in the
transplanted group. A new outer nuclear layer (ONL) was formed of a 4- or
5-fold, rhodopsin-positive photoreceptor cell layer.

[0118] (B). Immunostained human-specific mitochondria and rhodopsin in the
non-transplanted group of rd/SCID mice of the same age (8 weeks old)
which served as a control. Only a single outer nuclear layer was
observed, consisting mostly of cone photoreceptor cells. Almost no rod
photoreceptor cells were observed due to degeneration while only two
residual cells which were undergoing degeneration were detected.

[0119] (C). Immunostained human-specific mitochondria and recoverin in the
transplanted group. A 4- or 5-fold recoverin-positive cell layer formed a
new outer nuclear layer. In the transplanted group, a 4- or 5-fold
recoverin-positive cell layer was formed in the outer nuclear layer as
well as in the inner nuclear layer (INL).

[0120] (D). Immunostained human-specific mitochondria and recoverin in the
non-transplanted group used as a control. Positive responses were
detected in a total of 40 cells. A single recoverin-positive outer
nuclear layer consisted of cone-photoreceptor cells while the
recoverin-positive inner nuclear layer was formed of cone-bipolar cells.
[0121] Microscopic field: (A) and (C) Left. 200× magnification;
(A)-(D): 400× magnification. [0122] ONL: outer nuclear layer

[0123] INL: inner nuclear layer

[0124] RGC: retinal ganglion cell

[0125]FIG. 21 is a graph showing engraftment results after human
ESC-derived photoreceptor cells were transplanted into the mouse model of
retinal degeneration (rd/SCID).

[0126] In the non-transplanted group, rhodopsin was detected in only two
of a total of 199 cells per observation microscopic field (positive rate:
1.0%). On the other hand, 88 of a total of 215 cells per microscopic
field were rhodopsin positive in the transplanted group (positive rate:
40.8%) (p<0.0001). Accordingly, the transplanted rod photoreceptor
cells were found to occupy approximately 40% of the total area of the
retinal sections. Positive responses to recoverin were detected in 40 of
the total of 168 cells per microscopic field in the non-transplanted
group (positive rate: 23.8%), but in 120 of the total 292 cells per
microscopic field in the transplanted group (positive rate: 41.0%), with
statistical significance (p<0.0001).

[0127]FIG. 22 is of fluorescence microphotographs after the hESC-derived
photoreceptor cells had been transplanted into the mouse model of retinal
degeneration (rd/SCID).

[0128] Four weeks after the transplantation, human mitochondria and the
photoreceptor cell antigen synaptophysin were immunostained and analyzed.
In the non-transplanted group, recoverin means bipolar cells in the inner
nuclear layer and cone photoreceptor cells in the outer nuclear layer. In
the transplanted group, a 4- or 5-fold synaptophysin-positive cell layer
was found to form a new outer nuclear layer, suggesting that the
photoreceptor cells in the newly formed outer nuclear layer are in
synaptic interaction with other intraretinal cells within the retinas of
the transplanted mice. [0129] Microscopic field: Left. 200×
magnification; the others 400× magnification.

[0131]FIG. 24 is a graph showing positive rates of photoreceptor cell
markers versus the concentrations of Wnt3a and its inhibitor Dkk-1, in
the absence of Shh and retinoic acid (*: P<0.0001; **: P=0.0381)

BEST MODE

[0132] In accordance with an aspect thereof, the present invention
pertains to a composition for promoting the proliferation of retinal
cells, comprising a Wnt signaling pathway activator.

[0133] The term "Wnt signaling pathway activator", as used herein, is
intended to refer to a substance activating the Wnt signaling pathway
which has been found to regulate various processes during embryogenesis,
including cell-fate determination, reconstruction of organization,
polarity, morphology, adhesion and growth, and the maintenance and
proliferation of undifferentiated cells (Logan & Nusse, Annu Rev Cell Dev
Biol. 2004; 20: 781-810). As long as it transduces Wnt-mediated or
beta-catenin-mediated signals, any activator may be included within the
Wnt signaling pathway. The Wnt signaling pathway is a series of processes
that are initiated by the binding of the trigger Wnt to its receptor or
mediated by the stabilization of the downstream factor β-catenin.
The following is a description of how to activate Wnt signaling pathway.

[0135] 2) By increasing the level of β-catenin: most cells respond to
Wnt signaling pathway by an increase in the level of β-catenin. That
is, an increase in dephosphorylated β-catenin level or the
stabilization of β-catenin means the translocation of β-catenin
into the nucleus.

[0136] 3) By phosphorylation of dishevelled or phosphorylation of a
Wnt-accosiated receptor, LRP tail.

[0142] In a preferred embodiment of the present invention, the composition
for inducing the proliferation of retinal cells contains the Wnt
signaling pathway activator except for LiCl, BIO and SB415286 in an
amount of from 0.01 to 500 ng/ml, preferably in an amount of from 0.1 to
200 ng/ml, and more preferably in an amount of from 1 to 100 ng/ml. Among
the Wnt signaling pathway activators, LiCl is used in the medium in an
amount of 0.1 to 50 mM, preferably in an amount of 0.5 to 10 mM, and more
preferably in an amount of 1 to 10 mM; BIO is used in an amount of 0.1 to
50 μM, preferably in an amount of 0.1 to 10 μM, and more preferably
in an amount of 0.5 to 5 μM; SB415286 is used in an amount of 0.1 to
500 μM, preferably in an amount of 1 to 100 μM, and more preferably
in an amount of 5 to 50 μM. In a modification of the embodiment, the
medium may contain 50 ng/ml of Wnt3a or Wnt1; 50 or 100 ng/ml of Wnt5a
and Wnt11; 50 ng/ml of norrin; 2.5 or 5 mM of LiCl; 2 μM of BIO, or 30
μM of SB415286. The term "retina", as used herein, refers to
light-sensitive tissue. The retina is the innermost (sensory) transparent
coat in the eyeball and is directly relevant to vision. Just outside the
neurosensory retina is the retinal pigment epithelium consisting of
pigmented cells. In a broader sense, the retina includes the inner
sensory coat and the outer retinal pigmented epithelium. The retina is
located at the back of the eye and originates as outgrowths of the
developing brain in embryonic development. The retina is like a
five-layered cake, consisting of three nuclear layers with two network
layers intercalated therebetween. The three nuclear layers are: the
outermost nuclear layer consisting of photoreceptor cells; inner nuclear
layer consisting of horizontal cells, bipolar cells, amacrine cells, and
Muller glias; and the innermost retinal ganglion layer consisting of the
nuclei of retinal ganglion cells. After passing through the cornea and
the lens of the eye, light reaches the outer nuclear layer through the
retinal ganglion layer and the inner layer in that order, producing
neural impulses at photoreceptor cells. These neural impulses are
transduced in a reverse direction. That is, when photoreceptor cells are
stimulated by the neural impulses, nerve currents are transmitted to the
inner nuclear layer and then into optic nerve fibers through the retinal
ganglion cell layer.

[0143] As used herein, the term "retinal cell" is intended to refer to a
cell constituting a part of the retina, including photoreceptor cells
such as rod and cone photoreceptor cells, retinal ganglion cell,
horizontal cells, bipolar cells, amacrine cells, Muller glial cells, and
retinal pigmented epithelium. The composition of the present invention
induces retinal cells, particularly photoreceptor cells to proliferate.

[0144] As used herein, the term "photoreceptor cell" refers to a
specialized type of neuron found in the eye's retina that is capable of
phototransduction and allowing shapes and colors to be recognized: when
light reaches the retina through the cornea and the lens, the
photoreceptor cell converts the light energy into electric energy which
is then transmitted into the brain. There are two main types of
photoreceptor cells: rods and cones, which are adapted for low light and
bright light, respectively. Cone cells gradually become denser towards
the center of the retina, that is, the yellow spot, and function to
perceive images and colors while rod cells are distributed predominantly
at the periphery of the retina, allowing the perception of images and
light. Photoreceptor cells are characterized by the ability to express at
least one, two or three markers selected from among recoverin (rod
photoreceptor cells, cone photoreceptor cells), rhodopsin (rod
photoreceptor cells), peripherin2 (rod photoreceptor cells), rom1 (rod
photoreceptor cells, cone photoreceptor cells), Pde6b (rod photoreceptor
cells), arrestin sag (rod photoreceptor cells), phosducin (rod
photoreceptor cells, cone photoreceptor cells), synaptophysin (rod
photoreceptor cells, cone photoreceptor cells), red/green opsin (cone
photoreceptor cells), and blue opsin (cone photoreceptor cells).

[0145] In a preferred embodiment, the retinal cells or photoreceptor cells
of the present invention may be derived from animals including humans. As
used herein, the term "animal" is intended to include humans, primates,
cows, pigs, sheep, horses, dogs, mice, rats, and cats, humans being
preferred.

[0146] In an embodiment of the present invention, a Wnt signaling pathway
activator was identified as playing an important role in the
proliferation of retinal cells. In this regard, the Wnt signaling pathway
activator Wnt3a (W), the Shh signaling pathway activator Shh (S), and
retinoic acid (R) were combined with one another to afford various
compositions which were then applied to hESCs at different time points,
followed by culturing the cells for 29 days. After completion of the
differentiation, differentiated retinal cells were counted (Example 9).

[0147] The highest cell population was measured upon the use of W+/S+/R+
(3.98(±0.64)×106 cells), followed by
2.74(±0.36)×106 cells in W+/S-/R+ and
2.21(±0.67)×106 cells in W+/S+/R-. W-/S-/R- and W-/S+/R+
proliferated the cells at a density of as low as
0.87(±0.38)×106 and 0.73(±0.16)×106,
respectively (FIG. 23 and Table 8). The data indicate that cell
proliferation is independent of the presence of S and R, but depends on
W. A great part of the cell proliferation results from the effect of
Wnt3a. When comparing the two media W+/S+/R+ and W-/S+/R+, the cell
populations (3.98×106 vs. 0.73×106) differ by five
times (FIG. 23 and Table 8). This is another evidence that Wnt3a plays a
critical role in cell proliferation. After being cultured four weeks in
the (W+/S+/R+) culture for 4 weeks, the undifferentiated cells
proliferated to 3.98×106 differentiated cells (FIG. 23 and
Table 8), which was 257-fold higher than the population of the initial
cells, for the first time indicating that the Wnt signaling pathway
activator can be used for proliferating retinal cells.

[0148] As used herein, the term "stem cell" refers to a cell with
pluripotency to give rise to all derivatives of the three primary germ
layers (the endoderm, mesoderm and ectoderm) or one with multipotency to
differentiate into mature cells closely related in tissue type and
function.

[0149] As used herein, the term "animal" is intended to include humans,
primates, cows, pigs, sheep, horses, dogs, mice, rats, and cats, humans
being preferred.

[0150] As used herein, the term "embryonic stem cell" refers to a
pluripotent cell derived from the inner cell mass of the blastocyst
immediately before the nidation of a fertilized egg on the uterine wall,
which can differentiate into any type of animal cells, and is intended in
a broader sense to include stem cell-like cells such as embryoid bodies
and induced pluripotent stem (iPS) cells.

[0152] In accordance with another aspect, the present invention pertains
to a composition for inducing retinal progenitor cells to differentiate
into retinal cells, comprising a Wnt singling pathway activator.
Preferably, the retinal cells are photoreceptor cells.

[0153] The term "retinal progenitor cell", as used herein, is intended to
refer to a multipotent progenitor cell which can differentiate into cells
present in the retina and retinal pigmented epithelial cells. Retinal
progenitor cells may be cells which have been differentiated from various
stem cells, that is, retinal stem cells, cord blood stem cells, amniotic
fluid stem cells, fat stem cells, bone marrow stem cells, adipose stem
cells, neural stem cells, embryonic stem cells, induced pluripotent stem
cells, or somatic cells somatic cell nuclear transfer cells or may be
present within or isolated from the retina. A retinal progenitor cell
can, in general, undergo symmetric or asymmetric division and thus can
either differentiate into various types of retinal cells or retinal
pigmented epithelial cells, or produce two further retinal progenitor
cells. Thus, it should be understood that the cells that are used in the
culturing step to differentiate into retinal progenitor cells include
various types of cells which were generated during differentiation from
stem cells into retinal cells, as well as retinal progenitor cells.
Retinal progenitor cells include neural retinal progenitor cells and
retinal pigmented epithelial progenitor cells and are characterized by at
least one, two or three markers selected from among Rax, Pax6, Chx10,
Otx2, Sox2, Lhx2, Six3, Six6, and Mitf.

[0154] As used therein, the term "progenitor cell" or "precursor" refers
to a cell capable of asymmetric division. asymmetric division refers to
situations in which a progenitor cell or a precursor can, with a certain
probability, either produce two further progenitor or precursor cells or
differentiate, so that although they have undergone the same rounds of
passages, the resulting cells may have different ages and properties.

[0156] As used herein, the term "neural retinal progenitor cells" is
intended to mean the retinal progenitor cells which favor neurons. That
is, neural retinal progenitor cells are herein progenitor cells
determined to differentiate into intraretinal neurons (rod and cone
photoreceptor cells, retinal ganglion cells, horizontal cells, bipolar
cells, amacrine cells, and Muller glial cells). A neural retinal
progenitor cell can, in general, undergo symmetric or asymmetric
division, either differentiating into various types of retinal cells or
retinal pigmented epithelial cells, or producing two further retinal
progenitor cells. Thus, it should be understood that the cells in the
step of culturing that differentiate into neural retinal progenitor cells
include various types of cells generated during the differentiation of
stem cells into retinal cells as well as neural retinal progenitor cells.
Neural retinal progenitor cells are characterized by expressing at least
one, two or three markers selected from among Rax, Pax6, Chx10 and Crx.

[0157] In addition to expressing these markers, neural retinal progenitor
cells may be characterized by the ability to express Crx, recoverin and
rhodopsin, which are the markers of cells of the next differentiation
stage, that is, photoreceptor cell precursors and photoreceptor cells. On
the contrary, neural retinal progenitor cells are observed to have a
decreased expression level of Otx2, Sox2, Lhx2, Six3, Six6 and Mitf,
which are markers characteristic of retinal progenitor cells that
manifest themselves in the previous differentiation stage.

[0158] As used herein, the term "retinal pigmented epithelial progenitor
cell" is intended to refer to a differentiated retinal progenitor cell
which favors retinal pigmented epithelium. Retinal pigmented epithelial
progenitor cells are characterized by expressing one or more markers
selected from among Mift and Pax6.

[0159] In a preferred embodiment, differentiation from retinal progenitor
cells into retinal cells may be achieved by (a) culturing stem
cell-derived retinal progenitor cells in a composition containing a Wnt
signaling pathway activator to differentiate them into neural retinal
progenitor cells; (b) culturing the neural retinal progenitor cells in a
composition containing a Wnt signaling pathway activator to differentiate
them into photoreceptor cell precursors; and (c) culturing the
photoreceptor cell precursors in a composition containing a Wnt signaling
pathway activator to differentiate them into retinal cells including
photoreceptor cells.

[0160] As used herein, the term "photoreceptor cell precursor" means a
precursor favoring differentiation into a photoreceptor cell,
characterized by one or more markers selected from among Crx (rod and
cone photoreceptor cell precursors) and Nr1 (rod photoreceptor cell
precursors). In addition to expressing the markers, the photoreceptor
cell precursors may also be characterized by the ability to express at
least one, two or three of recoverin, rhodopsin, peripherin2, and rom1,
which are markers characteristic of differentiating photoreceptor cells.

[0162] In a preferred embodiment of the present invention, the composition
for inducing the proliferation of retinal cells contains the Wnt
signaling pathway activator except for LiCl, BIO and SB415286 in an
amount of from 0.01 to 500 ng/ml, preferably in an amount of from 0.1 to
200 ng/ml, and more preferably in an amount of from 1 to 100 ng/ml. Among
the Wnt signaling pathway activators, LiCl is used in the medium in an
amount of 0.1 to 50 mM, preferably in an amount of 0.5 to 10 mM, and more
preferably in an amount of 1 to 10 mM; BIO is used in an amount of 0.1 to
50 μM, preferably in an amount of 0.1 to 10 μM, and more preferably
in an amount of 0.5 to 5 μM; SB415286 is used in an amount of 0.1 to
500 μM, preferably in an amount of 1 to 100 μM, and more preferably
in an amount of 5 to 50 μM. In a modification of the embodiment, the
medium may contain 50 ng/ml of Wnt3a or Wnt1; 50 or 100 ng/ml of Wnt5a
and Wnt11; 50 ng/ml of norrin; 2.5 or 5 mM of LiCl; 2 μM of BIO, or 30
μM of SB415286. In a preferred embodiment, the composition for
inducing retinal progenitor cells to differentiate into neural retinal
progenitor cells in step (a) may further comprise an IGF1R activator, a
BMP signaling pathway inhibitor, and an FGF signaling pathway activator;
the composition for inducing retinal neural progenitor cells to
differentiate into photoreceptor cell precursors in step (b) may be free
of the BMP signaling pathway inhibitor and the FGF signaling pathway
activator and may further comprise an Shh (sonic hedgehog) signaling
pathway activator; and the composition for inducing the photoreceptor
cell precursors to differentiate into retinal cells including
photoreceptor cells in step (c) may be the same as in the composition of
step (b), with the exception that RA is further added thereto.

[0163] Any technique that is well known in the art or allows the
production of retinal progenitor cells may be employed to produce retinal
progenitor cells. Preferably, the retinal progenitor cells may be
obtained by: (a') culturing stem cells in a medium containing an IGF1R
activator, a Wnt signaling pathway inhibitor and a BMP signaling pathway
inhibitor to differentiate them into eye field precursors in the form of
floating aggregates; and (b') culturing the eye field precursors in the
form of floating aggregates in the same medium, but further comprising an
FGF signaling pathway activator to differentiate them into retinal
progenitor cells.

[0164] In a preferred embodiment, when cultured, the floating aggregates
of eye field precursors may be grown adhering to a plate. Any
cell-adhering plate well known in the art may be employed. Preferably, it
is coated with an extracellular matrix, such as poly-D-lysine, laminin,
poly-L-lysine, matrigel, agar, polyornithine, gelatin, collagen,
fibronectin or vitronectin. Most preferred is a plate coated with
poly-D-lysine/laminin. The cell population per floating aggregate which
adheres to the plate is the one that is the most highly efficient.
Preferably, a floating aggregate consists of 200-400 cells.

[0166] As used herein, the term "eye field precursor" refers to a cell
expressing a marker (eye field transcription factors; Zuber, et al.,
Development, 2003; 130: 5155-67) found in a progenitor for the eye field
of the forebrain neural plate. The eye field precursors are characterized
by at least one, two or three markers selected from among Six3, Rax,
Pax6, Otx2, Lhx2, and Six6.

[0167] As used herein, the term "floating aggregate" refers to a cell mass
floating in a medium which is generated when a floc of stem cells is
cultured for at least one day in a non-adhesive plate without feeding
mouse embryonic fibroblasts and sera. Depending on the composition of the
medium supplied, the eye field precursors may express eye field
transcription factors.

[0168] As used herein, the term "Wnt signaling pathway inhibitor" is
intended to refer to a factor which interrupts interaction between the
extracellular Wnt protein and the membrane protein Frizzled receptor or
LRP or inhibits intracellular Wnt-mediated signal transduction (Kawano &
Kypta, J Cell Sci. 2003; 116: 2627-34). So long as it inhibits
Wnt-mediated signal transduction, any Wnt signaling pathway inhibitor may
be used in the present invention. Examples of the Wnt signaling pathway
inhibitors useful in the present invention include the Dkk (Dickkopf)
family (Dkk-1, Dkk-2, Dkk-3 and Dkk-4), which are Wnt antagonists capable
of interacting with the co-receptor LRP, Wise, the sFRP (secreted
Frizzled-related protein) family, which functions as Wnt antagonists
binding to Wnt receptors, a Frizzled-CRD domain, WIF-1 (Wnt inhibitory
factor-1), IWP-2, IWP-3, IWP-4, cerberus, Wnt antibodies, dominant
negative Wnt proteins, overexpression of Axin, overexpression of GSK
(glycogen synthase kinase), dominant negative TCF, dominant negative
dishevelled and casein kinase inhibitors (CKI-7, D4476 etc.), with
preference for Dkk-1.

[0169] Wnt signal transduction may be inhibited by suppressing each
component involved in the Wnt pathway with for example RNAi, in addition
to the Wnt signaling pathway inhibitor.

[0170] In a preferred embodiment, IGF-1 or IGF-2 may be used as an IFG1R
activator, with priority given to IGF-2. The medium useful for inducing
the neural retinal progenitor cells to differentiate into photoreceptor
cell precursors contains IGF1R activator in an amount of from 0.01 to 100
ng/ml, preferably in an amount of from 0.1 to 50 ng/ml, more preferably
in an amount of from 1 to 20 ng/ml, and most preferably in an amount of
10 ng/ml.

[0171] In a preferred embodiment, the BMP signal pathway inhibitor include
noggin, chordin, twisted gastrulation (Tsg), cerberus, coco, gremlin,
PRDC, DAN, dante, follistatin, USAG-1, dorsomorphin and sclerostin, with
preference for noggin. The medium contains the BMP signaling pathway
inhibitor in an amount of from 0.01 to 100 ng/ml, preferably in an amount
of from 0.1 to 50 ng/ml, more preferably in an amount of from 0.5 to 20
ng/ml, and most preferably in an amount of 10 ng/ml.

[0172] As the FGF signaling pathway activator, a factor activating FGRR1c
or FGFR3c, FGF1, FGF2, FGF4, FGF8, FGF9, FGF17 or FGF19 may be used, with
FGF2 being preferred. The medium useful for inducing the retinal
progenitor cells to differentiate into neural retinal progenitor cells
contains the FGF signaling pathway activator in an amount of from 0.01 to
100 ng/ml, preferably in an amount of from 0.1 to 50 ng/ml, more
preferably in an amount of from 1 to 20 ng/ml, and most preferably in an
amount of 5 ng/ml.

[0173] In a preferred embodiment, examples of the Wnt signaling pathway
activator useful in the present invention include Wnt1, Wnt2, Wnt2b,
Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a,
Wnt9b, Wnt10a, Wnt10b, Wnt11, Wnt16b; substances increasing
β-catenin levels; GSK3 inhibitors such as lithium, LiCl, bivalent
zinc, BIO, SB216763, SB415286, CHIR99021, QS11 hydrate, TWS119,
Kenpaullone, alsterpaullone, indirubin-3'-oxime, TDZD-8 and Ro 31-8220
methanesulfonate salt; Axin inhibitors, APC inhibitors, norrin and
R-spondin 2, with preference for Wnt3a, Wnt1, Wnt5a, Wnt11, norrin, LiCl,
BIO and SB415286. The medium useful for inducing the neural retinal
progenitor cells to differentiate into photoreceptor cell precursors
contains the Wnt signaling pathway activator except for LiCl, BIO and
SB415286 in an amount of from 0.01 to 500 ng/ml, preferably in an amount
of from 0.1 to 200 ng/ml, and more preferably in an amount of from 1 to
100 ng/ml. Among the Wnt signaling pathway activators, LiCl is used in
the medium in an amount of 0.1 to 50 mM, preferably in an amount of 0.5
to 10 mM, and more preferably in an amount of 1 to 10 mM; BIO is used in
an amount of 0.1 to 50 μM, preferably in an amount of 0.1 to 10 μM,
and more preferably in an amount of 0.5 to 5 μM; SB415286 is used in
an amount of 0.1 to 500 μM, preferably in an amount of 1 to 100 μM,
and more preferably in an amount of 5 to 50 μM. In a modification of
the embodiment, the medium may contain 50 ng/ml of Wnt3a or Wnt1; 50 or
100 ng/ml of Wnt5a and Wnt11; 50 ng/ml of norrin; 2.5 or 5 mM of LiCl; 2
μM of BIO, or 30 μM of SB415286. In a preferred embodiment, the Shh
signaling pathway activator useful in the present invention may be Shh,
an inhibitor of Ptc's interaction with Smo, an Smo receptor activator, a
substance increasing Ci/Gli family levels, an inhibitor of the
intracellular degradation of Ci/Gli factors, or an Shh receptor activator
(e.g. Hg--Ag, purmorphamine, etc.). Preferred is Shh or purmorphamine. In
a preferred embodiment, the medium contains the Shh signaling pathway
activator in an amount of from 0.1 to 5,000 ng/ml, preferably in an
amount of from 1 to 2,500 ng/ml, more preferably in an amount of from 10
to 1,000 ng/ml, and most preferably in an amount of 250 ng/ml. In an
embodiment of the present invention, the medium contains Shh in an amount
of 250 ng/ml or purmorphamine in an amount of 1 μM.

[0174] As for the period of time for culturing after differentiation
starts, it is preferably given 1-30 days for step (a'), 5-15 days for
step (b'), 1-30 days for step (a), 1-30 days for step (b), and 1-60 days
for step (c), and most preferably 4 days for step (a'), 9 days for step
(b'), 5 days for step (a), 3 days for step (b), and 8-15 days for step
(c). In a preferred embodiment, it takes as short as approximately 29
days to complete the differentiation of stem cells into retinal cells,
allowing the method to be effectively applied to clinical treatment.

[0175] Differentiation into eye field precursors in step (a') may be
accomplished by inducing and promoting the development of the forebrain
during embryogenesis with the concomitant suppression of Wnt and BMP
signaling pathways (Piccolo, et al., Nature. 1999; 397: 707-10).
Therefore, the culture medium contains Dkk-1 as a Wnt signaling pathway
inhibitor, noggin as a BMP inhibitor, and IGF-1 as an IGF1R activator
functioning to promote the formation of the eye field in the forebrain.
Kinds and medium levels of the Wnt signaling pathway inhibitor, the BMP
inhibitor and IGF1R activator are as defined above.

[0176] The medium useful in step (a') contains IGF-1 at a concentration of
0.01-100 ng/ml, Dkk-1 at a concentration of 0.01-10,000 ng/ml and noggin
at a concentration of 0.01-100 ng/ml, and most preferably IGF-1 at a
concentration of 5 ng/ml, Dkk-1 at a concentration of 1 ng/ml, and noggin
at a concentration of 1 ng/ml.

[0178] Differentiation into retinal progenitor cells in step (b') may be
conducted in a medium containing an FGF signaling pathway activator,
preferably FGF2, in combination with the factors of step (a'), that is,
an IGF1R activator, a Wnt signaling pathway inhibitor and a BMP
inhibitor.

[0179] The medium useful in step (b') contains IGF-1 at a concentration of
0.01-100 ng/ml, Dkk-1 at a concentration of 0.01-10,000 ng/ml, noggin at
a concentration of 0.01-100 ng/ml and FGF2 at a concentration of 0.01-100
ng/ml and most preferably IGF-1 at a concentration of 10 ng/ml, Dkk-1 at
a concentration of 10 ng/ml, noggin at a concentration of 10 ng/ml and
FGF2 at a concentration of 5 ng/ml.

[0180] The differentiation of the retinal progenitor cells of step (b')
into neural retinal progenitor cells in step (a), must be conducted in
the absence of Dkk-1, a Wnt signaling pathway inhibitor, for there to be
a high level of Pax6 expressed (Pax6 is essential for the generation of
neural retinal progenitor cells), and in a medium containing Wnt3a for
promoting the Wnt pathway, noggin for converting ventral retinal
pigmented epithelium into the neural retina in the development stage of
optic vesicles and optic cups during embryogenesis, FGF2 for suppressing
the expression of genes necessary for retinal pigmented epithelium and
promoting the generation of neural retinal progenitor cells, and IGF-1
responsible for the antiapoptosis of the photoreceptor cells.

[0181] The medium useful in step (a) contains IGF-1 in a concentration of
0.01-100 ng/ml, noggin in a concentration of 0.01-100 ng/ml, FGF2 in a
concentration of 0.01-100 ng/ml, and Wnt3a in a concentration of 0.01-500
ng/ml and most preferably IGF-1 in a concentration of 10 ng/ml, noggin in
a concentration of 10 ng/ml, FGF2 at a concentration of 5 ng/ml and Wnt3a
at a concentration of 50 ng/ml.

[0182] Differentiation into photoreceptor cell precursors in step (b) is
conducted in a medium which is free of both noggin and FGF2 that
respectively inhibit Shh signaling pathway and Shh-induced rhodopsin
expression, and which contains IGF-1 for proliferating rod photoreceptor
cell precursors, Wnt3a for promoting the Wnt pathway, and Shh.

[0183] The medium useful in step (b) contains IGF-1 in a concentration of
0.01-100 ng/ml, Wnt3a at a concentration of 0.01-500 ng/ml and Shh at a
concentration of 0.1-5,000 ng/ml and most preferably IGF-1 at a
concentration of 10 ng/ml, Wnt3a at a concentration of 50 ng/ml and Shh
at a concentration of 250 ng/ml.

[0184] Differentiation into photoreceptor cells in step (c) is conducted
in a medium which contains RA (retinoic acid) for further promoting the
differentiation in combination with IGF-1, Wnt3a and Shh.

[0185] The medium useful in step (c) contains IGF-1 in a concentration of
0.01-100 ng/ml, Wnt3a at a concentration of 0.01-500 ng/ml, Shh at a
concentration of 0.01-5,000 ng/ml and RA at a concentration of 0.5-10,000
nM and most preferably IGF-1 at a concentration of 10 ng/ml, Wnt3a at a
concentration of 50 ng/ml, Shh at a concentration of 250 ng/ml and RA at
a concentration of 500 nM.

[0186] In steps (a) to (c), Wnt1, Wnt5a, Wnt11, norrin, LiCl, BIO or
SB415286 may be used instead of Wnt3a while purmorphamine may substitute
for Shh.

[0189] As a result, the photoreceptor cell markers Crx (p=0.0247),
recoverin (p=0.0113), rhodopsin (p=0.0166) and peripherin2 (p=0.0166)
remarkably decreased in expression level with increasing of Dkk-1
concentration. Statistical significances were found between all groups
except for group I vs. group II and group IV vs. group V in Crx and group
I vs. group II in recoverin (significance between groups: *: p<0.0001;
**: p=0.0381) (Table 10 and FIG. 24). These data showed that the
differentiation into photoreceptor cells is induced by a Wnt signaling
pathway activator and that the inhibition of the Wnt signaling pathway
leads to almost no generation of photoreceptor cells, giving evidence of
the Wnt signaling pathway inhibitor playing a critical role in
differentiation into photoreceptor cells.

[0190] On the other hand, respective substitutes may be used instead of
Wnt3a and Shh. In this context, the culturing processes are carried out
for the same time period under the same conditions with the exception
that 50 ng/ml of Wnt1; 50 and 100 ng/ml of Wnt5a and Wnt11; 50 ng/ml of
norrin; 2.5 or 5 mM of LiCl; 2 μM of BIO; or 30 μM of SB415286 is
used as a Wnt signaling pathway activator in substitution for Wnt3a and
purmorphamine is used as a substitute for Shh. These substitutes, as a
result, give rise to the differentiation and maturation of photoreceptor
cells in a similar pattern to that obtained with Wnt3a and Shh (Example
12).

MODE FOR INVENTION

[0191] A better understanding of the present invention may be obtained
through the following examples which are set forth to illustrate, but are
not to be construed as limiting the present invention.

[0194] While the medium was replaced with a fresh one every day, the
undifferentiated stem cells were passaged at a ratio of 1:9-1:15 every
six or seven days manually or with collagenase IV (Invitrogen), and then
transferred onto fresh MEF feeder cells. During the passage of the hESCs,
immunochemical staining with OCT-4 and SSEA-4 (Chemicon, Temecula,
Calif., USA), antigens characteristic of undifferentiated hESCs, was
conducted at regular intervals of time to monitor the degree of
differentiation. Cells that were found to have undergo differentiation
were removed.

[0195] The presence of mycoplasma contamination in the hESC culture, which
could have an undesirable effect on the differentiation of hESCs, was
regularly monitored with a kit (MycoAlert mycoplasma detection kit,
Lonza, Rockland, Me., USA).

[0198] While the medium was replaced with a fresh one every day, the
undifferentiated stem cells were passaged at a ratio of 1:4-1:6 every six
or seven days manually or with collagenase IV (Invitrogen), and then
transferred onto fresh MEF feeder cells. During the passage of the hESCs,
immunochemical staining with SSEA-4 (Chemicon, Temecula, Calif., USA) and
Nanog (abcam, Cambridge, Mass., USA), which are antigens characteristic
of undifferentiated human iPSCs, was conducted at regular intervals of
time to monitor the degree of differentiation. Cells that were identified
to have undergone differentiation were removed.

[0199] The presence of mycoplasma contamination in the hESC culture, which
could have an undesirable effect on the differentiation of hESCs, was
regularly monitored with a kit (MycoAlert mycoplasma detection kit,
Lonza, Rockland, Me., USA).

[0206] All the media were replaced with fresh ones every two or three days
in Examples 2 to 6, and the cells were cultured at 37° C. in a 5%
CO2 atmosphere. All induction and differentiation experiments were
repeated at least three times and the same results were obtained
therefrom.

[0207] The differentiation of the cells obtained in Examples 3 to 6 was
examined using an immunochemical staining method as follows.

[0208] The eye field precursors (floating aggregates) were cultured in
8-well poly-D-lysine/laminin-coated slides (BD Biosciences, Bedford,
Mass.) under the same conditions that were used for differentiation into
the retinal progenitor cells, the neural retinal progenitor cells, the
photoreceptor cell precursors and the photoreceptor cells. The cells
completely cultured in each step were fixed with 4% paraformaldehyde
(Sigma-Aldrich), after which non-specific reactions were blocked with PBS
containing 3% BSA (Jackson Immunoresearch Laboratory, Bar Harbor, Me.,
USA) and 0.25% Triton X-100 (Sigma-Aldrich).

[0210] Before use, these antibodies were diluted in a PBS solution
containing 1% BSA and 0.25% Triton X-100. The cells cultured on the
slides in each step were washed three times for 5 min with PBS and
incubated at room temperature for 2 hrs with species-specific secondary
antibody conjugated with Cy3 (1:800, Jackson Immunoresearch Laboratory)
or Alexa488 (1:500, Invitrogen). A standard material suitable for the
primary and the secondary antibody was used to examine non-specific
staining or interaction between the antibodies. Afterwards, the cells
were washed three times for 5 min with PBS, counterstained with DAPI
(4,6-diamidino-2 phenylindole) and mounted in Vectashield (Vector
Laboratories), followed by visualization under an epifluorescence
microscope (Nikon Eclipse, E800, Tokyo, Japan) and a confocal microscope
(Leica, Leica Microsystems Inc, Bannockburn, Ill., USA or Zeiss LSM510,
Carl Zeiss, Inc, Thornwood, N.Y., USA).

[0211] 500 cells were counted from 20 microscopic fields randomly selected
at 200 magnification and evaluated for positive responses to each
antibody. Positive responses to antibodies were determined after at least
three evaluations. Statistical analysis of the data was done using the
Kruskal-Wallis test and the Bland-Altman plot (Bland and Altman, 1986) of
Med Calc version 8.1.1.0 as well as the GEE (Generalized Estimating
Equations) model of SAS version 9.1. All data were represented as
mean±standard error of the mean (S.E.M) with a statistical
significance of p<0.05.

[0212] With regard to the markers characteristic of both forebrain eye
field precursors and retinal progenitor cells, Rax was found to be
expressed on the retinal progenitor cells generated in Example 3 at a
positive rate of 86.6±3.0%, Pax6 at a positive rate of 63.9±0.9%,
Otx2 at a positive rate of 76.4±2.0%, Sox2 at a positive rate of
83.0±1.9%, and Chx10 at a positive rate of 46.3±1.0%. The cells
were also found to express the marker Mitf characteristic of retinal
pigmented epithelial progenitor cells at a positive rate of 17.2±0.4%,
and the marker nestin characteristic of neural progenitor cells at a
positive rate of 65.7±2.7% (Tables 1 and 2).

[0213] On the neural retinal progenitor cells generated in Example 4, the
positive rates were increased simultaneously from 63.9% to 89.1% for Pax
6, from 86.6% to 98.2% for Rax, and from 46.3% to 64.5% for Chx10
(p<0.0001), indicating that the increase in the positive rate of Pax6
resulted from the proliferation of neural retinal progenitor cells (both
Rax+ and Pax6+ positive) (Tables 1 and 2).

[0214] As shown in Tables 1 and 2, the positive rate of the neural
progenitor cell marker nestin greatly decreased from 65.7% to 18.0%
(p<0.0001), from which it is understood that on differentiation day
18, the proliferation of neural progenitor cells was restrained while the
proliferation and differentiation of neural retinal cells were promoted.

[0215] On the other hand, the proliferative cell marker Ki67 rapidly
decreased from 87.5% to 31.5% (p<0.0001), indicating that
differentiation was more active than proliferation (FIG. 2 and Tables 3
and 4).

[0216] The positive rate of the photoreceptor cell precursor marker Crx
showed a drastic increase from 12.8% before Wnt3a addition to 80.1% after
Wnt3a addition (p<0.0001) (FIG. 2 and Tables 3 and 4). An increase in
the expression level from 35.8% to 68.5% (p<0.0001) was also found in
the universal photoreceptor cell marker recoverin, in the rod
photoreceptor cell marker rhodopsin from 35.3% to 52.9% (p<0.0001),
and in the photoreceptor cell's outer segment marker peripherin2 from
5.6% to 13.9% (p<0.0001) (FIG. 2, Tables 3 and 4). Mitf, an antigen
marker of early pigmented epithelial progenitor cells (Baumer, et al.,
Development. 2003; 130: 2903-15), showed a decrease in positive rate from
17.2% to 2.7% (p<0.0001) (Tables 1 and 2). Also, a decrease in
positive rate was found in the retinal progenitor cell markers Otx2
(p<0.0001) and Sox2 (p<0.0001), implying differentiation into
neural retinal progenitor cells.

[0217] In the photoreceptor cell precursors generated in Example 5,
decreased expression levels were detected for the markers of both retinal
progenitor cells and neural retinal progenitor cells, including Pax6
(from 89.1% to 61.6%, p<0.0001), Chx10 (from 64.5% to 48.5%,
p<0.0001) and Sox2 (from 68.1% to 49.1%, p<0.0001) (Tables 1 and
2). The photoreceptor cell precursor marker Crx also decreased from 80.1%
to 54.8% (p<0.0001) (FIG. 2, Tables 3 and 4). On the other hand, an
increase in expression level was detected in the photoreceptor cell
marker rhodopsin (from 52.9% to 60.5%, p=0.0489) and in the photoreceptor
cell's outer segment marker peripherin2 (from 13.9% to 26.0%,
p<0.0001), indicating the differentiation and maturation of
photoreceptor cells (FIG. 2, Tables 3 and 4). Further, the decreasing
positive rate of Ki67 was reversed into increasing from 31.5% to 58.4%
(p<0.0001) (FIG. 2, Tables 3 and 4), from which it is understood that
Shh started to promote cell proliferation.

[0218] As high as 82.4±4.6% of the population of cells generated by the
method of Example 6 for differentiation into photoreceptor cells were
found to react positive to recoverin, a universal marker of photoreceptor
cells (rod photoreceptor cells and cone photoreceptor cells), as measured
by a quantitative antigen assay (FIG. 3). Also, the quantitative antigen
assay showed that 81.2±2.5% of the cell population was positive to
rhodopsin, an antigen characteristic of rod photoreceptor cells (FIG. 3).
The fact that almost all of the rhodopsin-positive cells show a positive
reaction to recoverin ensures the reliability of the positive responses.

[0219] More precise determination of the existence and integrity of the
rhodopsin molecules formed by the method of the present invention was
carried out using human-specific recombinant rhodopsin (consisting of the
amino acids of the second extracellular loop in human rhodopsin) and
Ret-P1 (consisting of N-terminal amino acids at positions 4 to 10 in
rhodopsin), both of which are rhodopsin antibodies which recognize
different epitopes.

[0220] The human-specific recombinant rhodopsin and the Ret-P1 were found
to have identical positive rates, with a statistically significant
difference therebetween, as measured by the Bland-Altman plot (average of
difference in positive rate between the two antibodies: -1.00, 95%
confidence interval: -13.6-11.6). The cells reacted with almost no
difference in the positive rate with both human-specific recombinant
rhodopsin and Ret-P1 antibodies. This implies that the rhodopsin molecule
formed according to the method of the present invention retains two
different epitopes therein.

[0221] The rod photoreceptor cell's outer segment markers peripherin2
(Prph2) and rom1 (retinal outer segment membrane protein 1) were
positively detected in 41.2±2.0% and 76.0±4.6% of the population of
the cells cultured, respectively (FIG. 4). Both of these two markers were
observed only in rhodopsin-positive cells (FIG. 4). Positive rates in the
photoreceptor cells were measured to be 49.8±2.2% for phosducin, which
responds to light, that is, participates in the regulation of visual
phototransduction (FIG. 5) and 43.0±2.0% for synaptophysin which is
responsible for synaptic interactions with other intraretinal neurons
(FIG. 6). These facts prove that these cells participate in the neural
circuits of the retina and perform the function of transducing light
stimuli into neural electric stimuli. Taken together, the data obtained
above demonstrate that the method of the present invention can
differentiate with high efficiency human embryonic stem cells into
photoreceptor cells.

[0222] Blue opsin-cone photoreceptor cells were observed in 80.2±0.6%
of all the cells (FIG. 7). These cells were found from rhodopsin-positive
cell flocs or in the vicinity thereof, some of which were positive to
both rhodopsin and blue opsin. Accordingly, the method of the present
invention was proven capable of inducing human embryonic stem cells to
differentiate into both rod and cone photoreceptor cells.

[0223] Of the total population of the cells, 39.5±7.4% were positive to
Crx, a marker characteristic of photoreceptor cell precursors, and
30.2±6.1% were positive to Ki67, a nuclear marker of proliferating
cells (late G1 phase to M phase in the cell cycle). Photoreceptor cell
precursors are reported to express Crx immediately after leaving the cell
proliferation cycle. Also in the present invention, most of the human
ESC-derived, Crx-positive cells were observed to be negative to K167, a
marker associated with cell proliferation. However, the Crx-positive
cells were found to express Ki67 at a positive rate of 5.5±1.7% (FIG.
9).

[0224] From the data obtained above, it is apparent that the method of the
present invention can effectively differentiate photoreceptor cells from
human ESC-derived retinal progenitor cells in the same multi-step
induction pattern as they do from embryonic retinal progenitor cells,
where differentiation progresses in steps along the early and late
embryonic stage. The retinal progenitor cell marker Pax6 had a positive
rate of 89.7±1.9% and was detected in amacrine cells, which are a type
of differentiated retinal neurons. Meanwhile, the retinal progenitor
cells expressed Mitf (a marker of retinal pigmented epithelium (RPE)
progenitor cells) at a positive rate of 24.7±0.3%, and ZO-1 (a marker
of differentiated RPE) at a positive rate of 12.5±1.4%, and were also
positive to RPE65, a marker of differentiated RPE (FIG. 10).

[0225] To examine the differentiation of the photoreceptor cells generated
in Example 6, a reverse transcriptase-polymerase chain reaction (RT-PCR)
was performed on Day 29 of differentiation induction.

[0227] After initial DNA denaturation at 94° C. for 10 min, all PCR
was performed over 35 cycles starting with initial DNA denaturation
(94° C. for 30 sec, 60° C. for 30 sec, 72° C. for 1
min) and terminating with extension at 72° C. for 10 min. The PCR
products thus obtained were isolated by electrophoresis on 2% agarose gel
and analyzed.

[0228] All experiments were conducted in triplicate and human GAPDH was
used as a reference molecule for standard mRNA calculation.

[0229] The retinal progenitor cell-related genes RAX, PAX6, SIX3, SIX6,
LHX2 and Chx10 were detected in the RT-PCR products. Inter alia, RAX and
PAX6 were expressed to as the same high extent as was the quantitative
control gene GAPDH. Genes relevant to photoreceptor cells and other
retinal cells were also examined for mRNA expression. The PCR products
were observed to include the photoreceptor cell-associated genes CRX,
NRL, RCVRN, RHO, PDE6B, SAG and OPN1SW, the retinal ganglion cell-related
genes ATHO7 and POU4F2, and the amacrine cell-related gene NEUROD1, and
the bipolar cell-related gene ASCL1 (FIG. 16).

[0230] In order to investigate the differentiation of the photoreceptor
cells generated in Example 6, base sequencing was conducted on the
photoreceptor cell-specific genes recoverin (RCVRN: NM--002903.2)
and rhodopsin (RHO: NM--000539.3) on Day 29 of differentiation.

[0232] The RCVRN and RHO genes expressed in the photoreceptor cells
differentiated according to the present invention were found to perfectly
coincide with human standard sequences (http://www.ncbi.nlm.nih.gov),
indicating that the photoreceptor cells express human RCVRN and RHO genes
(FIG. 17).

Example 8

Transplantation of Photoreceptor Cells Differentiated from hESCs and
Evaluation of Transplanted Cells

<8-1> Transplantation of Differentiated Photoreceptor Cells

[0233] The transplantability and clinical applicability of the
photoreceptor cells generated in Example 6 to blind persons were examined
on the immunosuppressant retinal-degeneration mouse model rd/SCID.

[0234] After getting permission from the institutional Review Board of the
Seoul National University College of Medicine/the Seoul National
University Hospital and the Institutional Animal Care and Use Committee
(IACUC) of Seoul National University/the Seoul National University
Hospital, all animal experiments were conducted according to the ARVO
Statement for the Use of Animals in Ophthalmic and Vision Research. For
the transplantation, four-week-old C3H/Prkdc mice (rd/SCID, Jackson
Laboratory, BarHarbor, Me., USA) were employed. C3H/Prkdc mice were also
used as a negative control.

[0235] The retinal cells comprising photoreceptor cells differentiated
from hESCs according to the present invention were detached with accutase
and suspended in Dulbecco's PBS (D-PBS, Invitrogen) to form a single-cell
suspension with a density of 6-10×104 cells/μL. A surgical
procedure was carried out under a dissecting microscope (SZ51, Olympus,
Tokyo, Japan). After the mice were anesthetized by intrapeneal injection
of a mixture of zoletil (7.5 mg/kg, Virbac Laboratoires, Carros, France)
and xylazine (10 mg/kg, BayerKorea, Korea), drops of a
cycloplegic-mydriatic agent (Mydrin-P, Santen Pharmaceutical Co., Osaka,
Japan) and 0.5% proparacaine (Alcaine 0.5%, Alcon, Inc., Puurs, Belgium)
were put into the eyes. 1 μL of the cell suspension was transplanted
into each mouse by subretinal injection using a 10 μL injector
(NanoFil microsyringe, World Precision Instruments, WPI, Sarasota, Fla.,
USA) equipped with a 35-gauge needle (WPI).

[0236] The retinal function of the mice transplanted with the
photoreceptor cells differentiated from hESCs according to the present
invention was evaluated by electroretinography (ERG, Roland Consult,
Wiesbaden, Germany) 3-5 weeks after transplantation. For this, the mice
were acclimated to darkness for one day before the test. Prior to the
electroretinography, the mice were anesthetized by intraperitoneal
injection of a mixture of zoletil (7.5 mg/kg, Virbac Laboratoires,
Carros, France) and xylazine (10 mg/kg, BayerKorea, Korea) and drops of
Mydrin-P (Santen Pharmaceutical Co., Osaka, Japan) and 0.5% proparacaine
(Alcaine 0.5%, Alcon, Inc., Puurs, Belgium) were applied to the eyes.
Simultaneously, vidisic Gel (Dr. Mann Parma, Berlin, Germany) was also
put into the eyes so as to prevent eye dryness.

[0237] While the body temperature of the mice was maintained at 37°
C. on a mouse table (Roland Consult, Wiesbaden, Germany), the responses
of retinal nerves to light at intensities of -25, -10, and 0 dB were
recoded with the RETIport System (Roland Consult, Wiesbaden, Germany),
and the records were analyzed with the RETI-scan System (Roland Consult,
Wiesbaden, Germany). The same electroretinography was performed on
non-transplanted C57BL6 mice for positive control and on non-transplanted
C3H/Prkdc mice (rd/SCID) for negative control.

[0238] After the retinoelectrography, the mice were subjected to
euthanasia by CO2 injection and cervical dislocation. The eyeballs
were excised, followed by immunofluorescent staining. In detail, the
eyeballs excised immediately after euthanasia were fixed overnight at
4° C. with 4% paraformaldehyde, cryoprotected in 10% and 30%
sucrose (Sigma-Aldrich), embedded in OCT (Tissue-Tek, Sakura, Tokyo,
Japan), and immediately stored at -80° C. Four weeks after the
transplantation, some of the mice were subjected to euthanasia by
CO2 injection and cervical dislocation without electroretinography
before the excision of the eyeballs. Then, immunofluorescent staining was
performed on the eyeballs as described above.

[0239] Recovered signals in the electroretinograms reflect the effects and
efficacies of the cells transplanted into injured retinas. Particularly,
the existence of rhodopsin within the retina of the photoreceptor
cell-transplanted mice is closely associated with the amplitude of
b-waves. In the electroretinography, non-transplanted rd/SCID mice of the
same age (7-9 weeks old) did not respond to light stimuli (FIG. 18A). In
contrast, photoreceptor cell-transplanted rd/SCID mice gave definite
responses to light stimuli (FIG. 18B). The ERG b-wave from the eyes of
the photoreceptor cell-transplanted rd/SCID mice showed characteristic
wave forms (FIG. 18B) with an amplitude of 48.4(±3.4) μV (sample
size=13) (FIG. 19). Nowhere were characteristic wave forms found in the
ERG of the non-transplanted group, which showed a b-wave amplitude of
10.3 (±2.5) μV (sample size=17) different from that of the
transplanted group with statistical significance (p<0.0001) (Table 6,
FIG. 19).

[0240] The cryoprotected segments were sectioned at a thickness of 16
μm and fixed onto glass slides (Muto Pure Chemicals Co., Tokyo,
Japan), followed by immunochemical staining with antibodies to human
mitochondria and photoreceptor cells (no. of mouse models=13).

[0241] When the photoreceptor cells were transplanted into the retina of
the mice suffering from retinal degeneration, a new 4- or 5-fold
photoreceptor cell layer was formed, comprising the outer nuclear layer
consisting of cells characterized by rhodopsin and recoverin (FIG. 20).
There was a remarkable difference in the positive rate of rhodopsin and
recoverin between photoreceptor cell-transplanted group and
non-transplanted cells. In the non-transplanted group, rhodopsin was
detected in only two of a total of 199 cells per observation microscopic
field (positive rate: 1.0%). In contrast, 88 of a total of 215 cells per
microscopic field were rhodopsin positive in the transplanted group, with
an outer nuclear layer consisting of a 4- or 5-fold rhodopsin-positive
cell layer formed therein (positive rate: 40.8%) (p<0.0001) (Table 7
and FIG. 21).

[0242] As a result, the transplanted rod photoreceptor cells were found to
occupy approximately 40% of the total area of the retinal sections.
Rhodopsin discs, which are embedded in the membrane of the outer segment
of photoreceptor cells in the normal retina, were also found in the
photoreceptor cells of the transplanted mice (FIG. 20). Primarily
structured to convert light energy into electric energy, rhodopsin discs
are responsible for the first events in the perception of light. The
engrafted/differentiated photoreceptors of the transplanted mice,
particularly, the rhodopsin discs of the outer segment were thought to
include responses to light stimuli in the electroretinography, amplifying
the amplitude of the b-wave.

[0243] Recoverin expression was observed in 40 of the total of 168 cells
per microscopic field in the non-transplanted group (positive rate:
23.8%), but in 120 of the total 292 cells per microscopic field in the
transplanted group (positive rate: 41.0%) (p<0.0001) (Table 7 and FIG.
21). In the non-transplanted group, recoverin means bipolar cells in the
inner nuclear layer and cone photoreceptor cells in the outer nuclear
layer. In the transplanted group, a 4- or 5-fold recoverin-positive cell
layer was formed in the outer nuclear layer as well as in the inner
nuclear layer. From these observations, it is understood that when
transplanted into an injured retina, hESC-derived photoreceptor cells can
graft at proper loci onto the retina and effectively reconstruct the
outer nuclear layer which has been lost due to degeneration.

[0244] In addition, the engrafted cells were observed to express
synaptophysin, suggesting that they constructed, in synaptic interaction
with other intraretinal cells, retinal neurons and light circuits (FIG.
22). That is, the expression of synaptophysin indicates that the
photoreceptor cells in the newly formed outer nuclear layer are using
synapses to interact with other intraretinal cells to function as a
circuit in response to light.

[0245] Taken together, the data obtained above implies that when
transplanted, the photoreceptor cells differentiated from hESCs according
to the present invention can participate in the construction of retinal
circuits, exert their own functions in the transplanted subject, and open
up the new possibility of giving vision to patients suffering from
blindness that is attributed to the loss of photoreceptor cells.

[0246] In order to examine the role of a Wnt signaling pathway in
differentiation from retinal progenitor cells into retinal cells, various
combinations of three differentiation factors Wnt3a (W), Shh (S) and
retinoic acid (R) were used (FIG. 18 and Table 8). The differentiation
factors were added to media according to culture step and time. Wnt3a was
added at a concentration of 50 ng/ml on culture day 13, Shh at a
concentration of 250 ng/ml on culture day 18, and retinoic acid at a
concentration of 500 nM on culture day 21. In each medium, hESCs were
cultured for a total of 29 days before cell counting.

[0247] When undifferentiated hESCs were seed at a density of 1.3
(±0.1)×106 cell per well into 6-well cell culture plates,
303±12 floating aggregates were formed. For differentiation, 53 of the
floating aggregates (corresponding to 1.55×104
undifferentiated hESCs) were inoculated onto each well.

[0248] Statistical analysis of total cell populations in each medium was
done using the Kruskal-Wallis test, showing that the five media differed
in cell population from one to another with statistical significance
(p=0.0026). An additional analysis was conducted to examine differences
between groups. FDR-adjusted p-values for one error were obtained and are
summarized in Table 8, below.

[0249] The highest cell population was measured upon the use of W+/S+/R+
(3.98 (±0.64)×106 cells), followed by 2.74
(±0.36)×106 cells in W+/S-/R+ and 2.21
(±0.67)×106 cells in W+/S+/R-. W-/S-/R- and W-/S+/R+
proliferated the cells at a density of as low as 0.87
(±0.38)×106 and 0.73 (±0.16)×106,
respectively. The data indicate that cell proliferation is independent of
the presence of S and R, but depends on the presence of W and that a
great part of the cell proliferation results from the effect of Wnt3a.
When comparing the two media W+/S+/R+ and W-/S+/R+, the cell populations
(3.98×106 vs. 0.73×106) differ by five times. This
is another evidence that Wnt3a plays a critical role in cell
proliferation.

[0250] A statistical analysis showed that there was a difference between
W+/S+/R+ and W-/S+/R+ (p=0.03967), between W+/S+/R+ and W-/S-/R-
(p=0.03967), and between W-/S+/R+ and W+/S-/R+ (p=0.03967), with
significance (p<0.05) (FIG. 21). After being cultured four weeks in
the (W+/S+/R+) culture medium for 4 weeks, the undifferentiated cells
proliferated to 3.98×106 differentiated cells, which was
257-fold higher than the population of the initial cells.

Example 10

Role of Wnt Signaling Pathway Activator in Differentiation into Retinal
Cells

[0251] To differentiate the retinal progenitor cells into neural retinal
progenitor cells, Dkk-1 supply was quit on day 13 after induction and a
culture medium containing a combination of noggin, IGF-1, FGF2 and Wnt3a
(50 ng/ml) was supplied for 5 days. Wnt3a must be removed from the
culture media so as to induce and promote the development of the
forebrain and eye field precursors during the early embryogenesis.
However, Pax6-positive cells increased in a dose-dependent manner with
increase in Wnt3a concentration. Pax6 is expressed upon the development
of the retina during embryogenesis, that is, on all proliferating retinal
progenitor cells (Marquardt & Gruss. Trends Neurosci., 2002; 25: 32-8) in
addition to being essential for the generation of neural retinal
progenitor cells. Accordingly, an increased expression level of Pax6 is
indicative of highly efficient differentiation into neural retinal
progenitor cells. Wnt3a was supplied, with the concomitant removal of the
Wnt3a inhibitor, in order to induce the expression of Pax6 at a high
rate. The expression levels of retinal cell markers before and after
Wnt3a addition are given in Table 9, below.

[0252] On the differentiated neural retinal progenitor cells, the positive
rates were increased simultaneously from 63.9% to 89.1% for Pax 6
(p<0.0001), from 86.6% to 98.2% for Rax (p<0.0001), and from 46.3%
to 64.5% for Chx10 (p<0.0001), indicating that the increase in the
positive rate of Pax6 resulted from the proliferation of neural retinal
progenitor cells (both Rax+ and Pax6+ positive) (Table 9).

[0253] Nestin, a marker characteristic of most CNS neural progenitoc
cells, which is known to be not expressed in neural retinal progenitor
cells (Yang et al, Mech. Dev. 2000; 94: 287-91), was greatly decreased in
positive rate from 65.7% to 18.0% after Wnt3a addition (p<0.0001),
from which it is understood that the Wnt signaling pathway restrained the
proliferation of neural progenitor cells and promoted the proliferation
and differentiation of neural retinal cells.

[0255] With regard to the signal pathway for inducing differentiation from
retinal progenitor cells into Crx-positive photoreceptor cell precursors
in vivo, its exact biological changes and molecules have not yet been
reported in any prior art, so far (Ikeda et al., Proc. Natl. Acad. Sci.
USA, 2005; 102: 11331-6). In the present invention, however, Wnt3a was
first found to very strongly induce the expression of the photoreceptor
cell precursor marker Crx. That is, the positive rate of Crx showed a
drastic increase from 12.8% before Wnt3a addition to 80.1% after Wnt3a
addition (p<0.0001) (Table 9). The increased expression level of Crx
had on influence the differentiation and generation of photoreceptor
cells, directly promoting differentiation into photoreceptor cells. An
increased expression level was also found in the universal photoreceptor
cell marker recoverin (from 35.8% to 68.5%, p<0.0001), in the rod
photoreceptor cell marker rhodopsin (from 35.3% to 52.9%, p<0.0001),
and in the photoreceptor cell's outer segment marker peripherin2 (from
5.6% to 13.9%, p<0.0001) (Table 9)

Role of Inhibitor of Wnt Signaling Pathway Activator in Differentiation
into Retinal Cells

<11-1> Addition of Wnt Signaling Pathway Antagonist

[0257] In order to examine the function of the Wnt signaling pathway
activator on differentiation from hESCs into retinal cells, particularly
photoreceptor cells, cells were cultured in the presence of a Wnt
signaling pathway antagonist. The Wnt/Frizzled signaling pathway
inhibitor Dkk-1 (R&D Systems, Minneapolis, Minn., USA) was added at a
concentration of 10 ng/ml, 100 ng/ml or 1 μg/ml on day 13, followed by
culturing the cells for an additional 16 days. While the antagonist
concentrations were maintained uniformly over the culture time period,
the Wnt signaling pathway activator was assayed for activity. Dkk-1 was
dissolved in 0.1% bovine serum albumin (BSA, Sigma-Aldrich) in PBS before
use.

[0258] According to a protocol for differentiation into retinal cells,
Wnt3a and Dkk-1 were added on Day 13 at the following concentrations: 50
ng/ml Wnt3a (group I), free of both Wnt3a and Dkk-1 (group II), 10 ng/ml
Dkk-1 (group III), 100 ng/ml Dkk-1 (group IV), and 1 μ/ml Dkk-1 (group
V). In each medium, the cells were cultured to Day 29. In this
experiment, none of the other major differentiation factors Shh and RA
were used.

<11-2> Immunofluorescent Staning

[0259] To examine the effect of the Wnt signaling pathway activator on the
cell differentiation of Example 11-1, an immunofluorescent staining
analysis was performed as follows. On Day 29, the cells which had been
cultured on poly-D-lysine/laminin-coated, 8-well slides (BD Biosciences)
under the conditions of Wnt3a and Dkk-1 (groups I-V) were fixed with 4%
paraformaldehyde (Sigma-Aldrich), after which non-specific reactions were
blocked with PBS containing 3% BSA (Jackson Immunoresearch Laboratory,
Bar Harbor, Me., USA) and 0.25% Triton X-100 (Sigma-Aldrich).

[0261] Before use, these antibodies were diluted in a PBS solution
containing 1% BSA and 0.25% Triton X-100. The cells were washed three
times for 5 min with PBS and incubated at room temperature for 2 hrs with
species-specific secondary antibody conjugated with Cy3 (1:800, Jackson
Immunoresearch Laboratory) or Alexa488 (1:500, Invitrogen). A standard
material suitable for the primary and the secondary antibody was used to
examine non-specific staining or interaction between the antibodies.
Afterwards, the cells were washed three times for 5 min with PBS,
counterstained with DAPI (4,6-diamidino-2 phenylindole) and mounted in
Vectashield (Vector Laboratories), followed by visualization under an
epifluorescence microscope (Nikon Eclipse, E800, Tokyo, Japan) and a
confocal microscope (Leica, Leica Microsystems Inc, Bannockburn, Ill.,
USA or Zeiss LSM510, Carl Zeiss, Inc, Thornwood, N.Y., USA).

[0262] 500 cells were counted from 20 microscopic fields randomly selected
at 400× magnification and evaluated for positive responses to each
antibody. Positive responses to antibodies were determined after at least
three evaluations. After the data was corrected for cluster effects,
statistical analysis of the data was done using the GEE (Generalized
Estimating Equations) model of SAS version 9.1 so as to investigate
changes in positive rate with concentrations of Wnt3a and Dkk-1. All data
were represented as mean±standard error of the mean (S.E.M) with a
statistical significance of p<0.05.

[0263] As a result, significant differences were detected among the
photoreceptor cell marker. The photoreceptor cell markers Crx (p=0.0247),
recoverin (p=0.0113), rhodopsin (p=0.0166) and peripherin2 (p=0.0166)
remarkably decreased in expression level with increasing of Dkk-1
concentration. Statistical significances were found between all groups
except for group I vs. group II and group IV vs. group V in Crx and group
I vs. group II in recoverin (significance between groups: *: p<0.0001;
**: p=0.0381) (Tables 10 and 11 and FIG. 24). These data showed that the
differentiated photoreceptor cells resulted from the activation of the
Wnt signaling pathway and that the inhibition of the Wnt signaling
pathway led to almost no generation of photoreceptor cells.

[0264] The rhodopsin which was expressed at a positive rate of 11.9% in
spite of 1 μg/ml Dkk-1 was thought to be attributed to the
stabilization of beta-catenin by a Wnt independent mechanism or the
activation of non-canonical pathway by Wnt, Wnt5 and Wnt11. Also, the
expression level of the retinal progenitor cell marker Pax6 significantly
differed from one group to another over the overall 5 groups (p=0.0275)
and was decreased with an increase in, Dkk-1 concentration (all p values
between groups: p<0.0001) (FIG. 24, Tables 9 and 10).

[0266] The cells differentiated with various substitutes for Wnt3a and Shh
were subjected to immunochemical staining in the same manner as in
Example 7-1 and assayed for positive rates of retinal cell markers, and
the results are summarized in Tables 12 and 13, below.

[0267] As is apparent from the data of Tables 12 and 13, the Wnt3a
substitutes, Wnt1, Wnt5a, Wnt11, norrin, LiCl, BIO and SB415286 and the
Shh substitute purmorphamine allowed the retinal cell markers to be
expressed at similar positive rates to those when Wnt3a and Shh were
used.